1. Field of the Invention
The present invention relates generally to the field of biology. More particularly, it relates to cell-derived extracellular matrices and uses of the same.
2. Description of the Related Art
Stem cells are one of the most fascinating areas of biomedicine today and hold great promise as a means to increase the healthy life-span of an aging worldwide population. The great promise of stem cells is due in large part to the tremendous plasticity and immaturity of human embryonic stem cells (hES cells) and the viral vector engineered cousins known as induced pluripotent stem cells (iPS cells). However, critical unsolved issues impair their therapeutic potential. For example, maintenance of hES cells requires the use of mouse embryonic feeder cells to inhibit their differentiation. This practice has the potential to cause mouse-to-human pathogen transfer referred to as “xenorisk.” Additionally, and aside from the controversial human embryo-source of hES cells, the phenomenal plasticity and self-renewal capability of natural ES cells and the uncertainties associated with the use of viral vectors for iPS cells may yield an under-appreciated disadvantage: the uncertain reliability and predictability of these cells in clinical applications, especially over the long-term.
As stem cells, mesenchymal stem cells (MSCs) are characterized by their ability to both self-renew and to differentiate into specific cell types in response to appropriate lineage-specific growth factors, for example, to differentiate into osteoblasts on stimulation with BMP-2. Examples of cell types that MSCs may differentiate into include, but are not limited to, osteoblasts, stromal cells that support hematopoiesis and osteoclastogenesis, chondrocytes, myocytes, adipocytes, neuronal cells, endothelial cells, and β-pancreatic islet cells (Prockop, 1997; Dennis et al., 1999; Ferrari et al., 1998). Moreover, MSCs are ideally suited for cell-based tissue engineering, for example, for the repair of skeletal tissue in nonunion fractures and reconstructive surgery (Muschler et al., 2004).
When MSCs divide, there are three possible fates (FIG. 13). Stem cells may divide asymmetrically to give a daughter stem cell and a more differentiated progeny, or symmetrically to give either two identical daughter stem cells or two more differentiated cells. As a result of these processes, MSCs produce new mature cells, such as osteoblasts, throughout life via orchestration of stem cell self renewal, together with the regulated expansion of early transit amplifying progenitors (uncommitted progenitors) and subsequent commitment to a particular lineage (Loeffler and Potten, 1997; Aubin and Triffitt, 2002). Regulation of these events allows preservation of stem cells, expansion of stem cells, and production of differentiated progeny when needed for tissue repair. Because of these capabilities, MSCs are involved in tissue regeneration throughout life. However, relatively little is known about the cellular and molecular mechanisms underlying the control of mesenchymal stem cell (MSC) proliferation, differentiation, and survival. This presents difficulties in following and characterizing cells along the lineage because of the inability to isolate and obtain a sufficient number of homogeneous MSCs using current culture systems for in vitro expansion.
MSCs are of great therapeutic potential due to their capacity of self-renewal and multilineage differentiation and have been proposed for treatment of degenerative diseases such as osteoarthritis and osteoporosis, of children with osteogenesis imperfecta (Horwitz et al., 2002; Kassem, 2006; Banerjee and Bhonde, 2007), for promoting healing of nonunion fractures (Petite et al., 2000), and for enhancing reconstitution of hematopoietic and immune systems after marrow ablation by chemotherapy or radiotherapy for treatment of leukemia and related diseases (Koc et al., 2000). However, lack of information on the factors that control MSC behavior has made implementation of such therapeutic strategies difficult.
Another major bottleneck in clinical application of MSCs has been their limited number, because they are rare in the primary tissue (approximately 0.001%) (Wexler et al., 2003). Earlier attempts to expand the MSCs ex vivo from rodent or human marrow have proven difficult. Adjusting the cellular machinery to allow greater proliferation can lead to other unwanted outcomes, such as unmanageable precancerous changes, or differentiation down an undesired pathway. Moreover, MSCs tend to lose their stem cell properties under traditional cell culture conditions. This situation has impaired the use of MSCs for practical purposes, such as therapeutic purposes.
When cultured on traditional tissue culture plastic systems, MSCs tend to lose their ability to self-renew and instead undergo senescence or “spontaneously” differentiate into osteoblastic cells, stromal cells, and adipocytes (DiGirolamo et al., 1999; Banfi et al., 2000; Baksh et al., 2004; Izadpanah et al., 2008; Kim et al., 2009). Furthermore, with extensive passaging, the stem cell population is likely diluted by the generation of more committed, transiently amplifying and differentiated cells and the MSCs often lose multilineage differentiation potential (Banfi et al., 2000; Baksh et al., 2004; Izadpanah et al., 2008; Kim et al., 2009). This suggests that the principal fate of MSCs is self-renewal without amplification and/or differentiation when cultured under these conditions, indicating that a critical factor(s) present in the marrow microenvironment responsible for the maintenance of MSC properties (stemness) is missing in such “standard” culture systems. In fact, loss of stem cell properties and “spontaneous” differentiation when MSCs are cultured on plastic may actually represent the response of MSCs to growth factors produced endogenously in these cultures. These problems have impaired efforts to expand MSCs in culture for the purpose of studying molecular mechanisms that govern self-renewal and differentiation and for investigating their potential therapeutic use (Baksh et al., 2004).
Several approaches have been used in an attempt to preserve the properties of MSCs. The use of surface markers or differential adhesion strategies to enrich MSCs prior to expansion on tissue culture plastic has not been successful. Cultures with specific growth factor cocktails, such as fibroblast growth factor and leukemia inhibitory factor, have generally failed because the growth factors inevitably favor a particular lineage and cause loss of self-renewal capacity and multipotentiality (Jiang et al., 2002; Bianchi et al., 2003; Sotiropoulou et al., 2006).
Other previous attempts to restrain “spontaneous” MSC differentiation have involved culture on fibronectin matrices under low oxygen tension (3-5%) (D'Ippolito et al., 2006) to mimic the microenvironment of the bone marrow (Chow et al., 2001) or cultures at low seeding density in low serum in the presence of growth factors (Sekiya et al., 2002; Peister et al., 2004). These conditions permitted expansion of mouse and human MSCs for as many as 60 population doublings, but the full differentiation potential and cellular composition of these cell preparations remain unclear. Particularly, the ability of such cell preparations to form skeletal tissue in vivo has not been reported. Although introduction of telomerase into stem cells (Gronthos et al., 2003) or four transcription factor genes (Oct4, Sox2, c-myc, and Klf4) into somatic cells to reprogram these cells to pluripotent stem cells has been successful (Takahashi et al., 2007; Yu et al., 2007), this procedure alters cell behavior via genetic modification, making these cells unpredictable for use in human therapy. Specifically, retroviruses used to trigger the reprogramming process can disrupt the normal function of DNA and the development of tumor formation (Okita et al., 2007). In addition, fibroblast growth factor (FGF)-2 has been reported to increase the size of human MSC colonies and to restrain their differentiation, but FGF-2 reduced colony number (Bianchi et al., 2003). Other investigators have reported that FGF-2 alters the properties of human MSCs and may even enhance osteoblastogenesis while reducing neurogenic capability (Sotiropoulou et al., 2006). It has also been reported that expansion of human and mouse MSCs is accompanied by cellular senescence and outgrowth of transformed cells, though transformation is less frequent in cultured human MSCs (DiGirolamo et al., 1999; Rubio et al., 2005; Miura et al., 2006; Rosland et al., 2009; Ksiazek, 2009).
Therefore, there remains a need for methods and compositions that provide for the maintenance, expansion, and use of stem cells.